There is a plethora of literature relating to the expression of heterologous genes in lower organisms such as unicellular bacteria, yeast and filamentous fungi, and in higher cell types such as mammalian cells. There are also numerous reports on the production of transgenic animals, most of which relate to the production of transgenic mice. See, e.g., U.S. Pat. No. 4,736,866 (transgenic mice containing activated oncogene); Andres, A., et al. (1987) Proc. Natl. Acad. Sci. USA 84:1299-1303 (HA-RAS oncogene under control of whey acid protein promoter); Schoenberger, C. A., et al. (1987) Experientia 43:644 and (1988) EMBO J. 7:169-175 (C-myc oncogene under control of whey acid protein promoter); and Muller, W. J., et al. (1988) Cell 54:105-115 (C-myc oncogene under control of the mouse mammary tumor virus promoter). Several laboratories have also reported the production of transgenic Porcine species (Miller, K. F., et al. (1989) J. Endocrin. 120:481-488 (expression of human or bovine growth hormone gene in transgenic swine); Vize, P.D., et al. (1988) J. Cell Sci. 90:295-300 (porcine growth hormone fusion gene in transgenic pigs); and Ebert, K. et al. (1988) Mol. Endocrin. 2:277-283 (MMLV-rat somatotropin fusion gene in transgenic pigs)), transgenic sheep (Nancarrow, et al. (1987) Theriogenology 27:263 (transgenic sheep containing bovine growth hormone gene) Clark, A. J. et al. (1989) Bio/Technology 7:487-482 and Simons, J., et al. (1988) Bio/Technology 6:179-183 (human factor IX and .alpha.-1 antitrypsin CONA in bovine species), and rabbit (Hanover, S. V., et al. (1987) Deutche Tierarztliche Wochenschrift 94,:476-478 (production of transgenic rabbits by injection of uteroglobin-promoter-CAT fusion gene into fertilized rabbit oocytes). A number of reports have also suggested the production of transgenic cattle (Wagner, et al. (1984) Theriogenology 21:29-44) with one reporting some progress in microinjection techniques (Lohse, J. K., et al. (1985) Theriogenology 23:205). However, little, if any, success has been achieved in producing transgenic cows. Scientific articles which clearly demonstrate the actual production of a transgenic cow capable of producing a heterologous protein are presently unknown. This, despite the statements that one transgenic cow was produced in Canada which expressed human .beta.-interferon (Van Brunt, J. (1988) Bio/Technology 6:1149-1155) and that transient expression of human .alpha.-fetoprotein in liver and blood was obtained on one occasion (Church, R. B. (1986) Biotechnology News Watch 6 (15), 4). One reference reports that bovine papilloma virus was apparently integrated but not expressed in a transgenic cow (Roschlau, et al. (1988) Arch. Tierz., Berlin 31:3-8). A recent article has summarized the genetic engineering of livestock. (Pursel, V. G. et al. (1989) Science 244:1281-1288).
A number of laboratories have reported tissue-specific expression of DNA encoding various proteins in the mammary gland or the production of various proteins in the milk of transgenic mice and sheep. For example, Simmons, J. P., et al. (1987) Nature 328:530-532 report the microinjection of a 16.2 kb genomic fragment encoding .beta.-lactoglobulin (BLG) including 4 kb of 5' sequence, 4.9 kb of the BLG transcription unit and 7.3 kb of 3' flanking sequence into fertilized mouse eggs. According to these authors, the sheep BLG was expressed in mammary tissue and produced BLG in the milk of the transgenic mice at concentrations ranging from about 3.0 to about 23 mg/ml. When, however, cDNA encoding human factor IX or human al-antitrypsin was inserted into the 5' untranslated region of the BLG gene and microinjected into sheep (Simmons, J. P., et al. (1988) Bio/Technology 6:179-183) the production of factor IX or .alpha.1-antitrypsin was significantly reduced (25 ng/ml for factor IX and lomg/ml for .alpha.1-antitrypsin; see Clark, A. J., et al. (1989) Bio/Technology 7:487-492).
In a similar approach, a 14 kb genomic clone containing the entire 7.5 kb rat .beta.-casein together with 3.5 kb of 5' and 3.0 kb of 3' flanking DNA was reportedly microinjected into fertilized mouse oocytes. Lee, et al. (1988) Nucl. Acids Res. 16:1027-1041. Yet, in this case, the level of expression of the rat .beta.-transgene in the lactating mammary gland of transgenic mice was reported to be at a level of 0.01-1% of the endogenous mouse .beta.-casein gene.
Human tissue plasminogen activator (t-PA) reportedly was produced in transgenic mouse milk at the levels between 0.2 and about 0.4 .mu.g/ml when a cDNA encoding a human t-PA with its endogenous secretion sequence was expressed under control of a 2.6 kb 5' sequence of the murine whey acid protein gene. Gordon, K., et al. (1987) Bio/Technology 5:1183-1187. Subsequent experiments using the same or similar construction reportedly produced t-PA in different mouse lines arranging from less than 20 ng of t-PA per ml of milk to about 50 .mu.g/ml. Pittius, C. W., et al. (1988) Proc. Natl. Acad. Sci. USA 85:5874-5878.
U.S. Pat. No. 4,873,316 issued Oct. 10, 1989, discloses the use of 9 kb of 5' sequence from the bovine .alpha.S1 casein gene including the casein signal peptide and several casein codons fused to a mature t-PA sequence. The transgenic mice obtained with this construct reportedly produced about 0.2-0.5 .mu.g/ml of a t-PA fusion protein in their milk.
In addition, a number of patent publications purportedly describe the production of specific proteins in the milk of transgenic mice and sheep. See, e.g. European Patent Publication No. 0 264 166 published Apr. 20, 1988 (hepatitis B surface antigen and t-PA genes under control of the whey acid promoter protein for mammary tissue specific expression in mice); PCT Publication No. WO88/00239 published Jan. 14, 1988 (tissue specific expression of a transgene encoding factor IX under control of a whey protein promoter in sheep); PCT Publication No. WO88/01648 published Mar. 10, 1988 (transgenic mouse having mammary secretory cells incorporating a recombinant expression system comprising a bovine .alpha.-lactalbumin gene fused to interleukin-2); European Pat. Pub. No. 0 279 582 published Aug. 24, 1988 (tissue-specific expression of chloramphenicol acetyltransferase under control of rat .beta.-casein promoter in transgenic mice); and PCT Pub. No. WO88/10118 published Dec. 29, 1988 (transgenic mice and sheep containing transgene encoding bovine aSi casein promoter and signal sequence fused to t-PA).
Given the state of the transgenic art, it is apparent that a need exists for methods which enable the efficient production of transgenic mammals, especially transgenic mammals other than transgenic mice.
Further, it is apparent that a need exists for methods for producing transgenic bovine species which are capable of producing recombinant polypeptides such as human milk proteins and human serum proteins in the milk of such transgenic mammals.
Accordingly, it is an object herein to provide methods for detecting the transgenesis of fertilized oocytes prior to implantation.
In addition, it is an object herein to provide transgenic bovine species which are capable of producing recombinant polypeptides which are maintained intracellularly or are secreted extracellularly.
It is also an object herein to provide transgenic bovine species which are capable of producing recombinant polypeptides such as human milk proteins and human serum proteins in the milk of such transgenic animals.
Further, it is an object herein to provide milk from a transgenic bovine species containing such recombinant polypeptides.
Still further, it is an object herein to provide food formulations supplemented with recombinant polypeptides from such transgenic milk such as human infant formula supplemented with human lactoferrin.
Further, it is an object herein to provide transgenes which are capable of directing the production of recombinant polypeptides in the milk of transgenic bovine species.
The references discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by priority based on earlier filed applications.